The goal of this interagency agreement is to provide support of National Toxicology Program (NTP) hazard identification (or hazard assessment) activities targeted toward the prevention of diseases or adverse effects caused by environmental exposure to chemical or physical agents. These cooperative studies continue to improve the risk assessment process by determining quantitatively what constitutes an adverse health effect on the immune system in humans. These studies evaluate unique cohorts of individuals from professions associated with immune-mediated occupational diseases including asthma, respiratory and contact allergy, chronic beryllium disease, rhinitis, and silicosis. Occupational cohorts are being studied for a number of endpoints including, impact of genetic polymorphisms on inflammatory disease development and clinical outcomes, the role that genetic variations play in environmental and workplace related diseases and identification of unique immunological biomarkers for disease. Occupational rhinitis is a chronic inflammatory disease of the upper respiratory tract that is observed in greater than 90% of workers with occupational asthma. A cohort of chronic rhinosinusitis patients is being evaluated for subjects with allergies to molds. Patient recruitment and testing has been completed. Data has been collected and statistically analyzed. Analysis of allergic sensitization has shown that patients with chronic rhinosinusitis are more likely to be sensitized to a variety of environmental aeroallergens. The results derived from this study have provided further insight into the role of allergic sensitization in chronic rhinosinusitis. To investigate the diversity of fungi in the indoor built environment, air and dust samples have been collected from 30 homes of asthmatic children in the Kansas City area, and are currently in storage. Efforts have focused on developing an effective DNA isolation technique, both in terms of reducing bias against smaller-spore species, and in terms of sensitivity. Isolation of DNA from the Kansas City air and dust samples, amplifying and sequencing the fungal ribosomal internal transcribed spacer (ITS) regions, and assessing the diversity of fungi within these samples has begun. The results will then be incorporated into the health and environmental data of the homes to examine a possible correlation between the fungi present and the health of the inhabitants. In addition, a polymerase chain reaction (PCR) primer/probe set for the detection of toxigenic fungi within the Chaetomium genus is being developed. Species within the Chaetomium genus are often associated with moisture infiltration, and their presence can be an indication of mold problems. A Chaetomium globosum gene that shares strong similarity to the Che a chaetoglobosin (mycotoxin) biosynthesis gene in Penicillium expansum has been identified and a primer/probe set was developed to target this gene and tested against 13 other common environmental fungi. To date, the data indicate that this primer/probe set is specific to C. globosum, and it currently being evaluated against C. strumarium and C. atrobrunneum (two Chaetomium species implicated in clinical mycoses). Patient recruitment and serological sampling has been completed for an evaluation of mold allergy in 677 adult male farmers from the Agricultural Health Study, a cohort of pesticide applicators from North Carolina and Iowa. All sera have been analyzed for both total immunoglobulin E and specific IgE to molds using a mixture of mold antigens. Preliminary results suggest that agricultural practices may be associated with mold sensitization and may differ from those associated with atopy. Work is progressing in assessing an amine probe (pyridoxylamine, PYDA) for both identification and potential potency assessment of electrophilic contact allergens by kinetic spectrophotometric analyses. To date, 24 known sensitizers and 4 non-sensitizers have been assessed. This methodology has been set up at Portland State University Chemistry Department where stopped flow spectrophotometry for assessment of initial kinetic and very rapidly reacting allergens is being assessed. A reactivity constant (Ka) is determined for each allergen and compared to published values for sensitization. A series of benzoquinones is initially being assessed to identify structural activity relationships to binding, and mechanism(s) rates of binding. Work is planned on evaluating the potential incorporating less polar solvents to allow assessment of poorly soluble allergens and to expand the library of allergen Ka[unreadable]s for binding to both the amine and thiol spectrophotometric probes. Finally, ongoing studies are identifying and quantifying the volatile reaction products of gas-phase compounds present in the indoor environment, especially dicarbonyls, and investigating immunotoxic and hypersensitivity effects of these reaction products in both in vitro and in vivo models. Studies to characterize the effects of dicarbonyls on the respiratory tract have been conducted using an in vitro indoor air exposure system. Changes in inflammatory cytokine expression were evaluated in a pulmonary epithelial cell line (A549) after exposure to (i) a mixture of limonene and ozone, (ii) air contaminated only with limonene, and (iii) clean air. Preliminary data indicate that exposure to limonene was more inflammatory than exposure to clean air and a mixture of limonene and ozone was more inflammatory than exposure to limonene alone. Additional experiments are underway using different chemical concentrations and time points to confirm these findings. The indoor air contaminant 4-oxopentanal (4-OPA) was examined for irritancy and hypersensitivity following both dermal and pulmonary exposure using a murine model. 4-OPA was tested in a combined local lymph node assay and identified to be an irritant and strong sensitizer (EC3=0.08%) with a delayed hypersensitivity response supported by a positive ear-swelling test. Pulmonary exposure to 4-OPA caused a significant elevation in nonspecific airway hyperreactivity, with increased numbers of lung associated lymphocytes and neutrophils, and increased interferon-g production by lung associated lymph nodes. Further investigation into the role of structurally similar indoor air chemicals present in mixtures in the indoor environment and identification of conserved chemical structures such as di-oxygenated organic compounds among indoor air reaction products that may affect immunotoxic potential is ongoing. Improved chemical derivatization and gas chromatography/mass spectrometry techniques will be used for sampling of indoor air immunotoxic compounds.